The instant invention relates to the field of protein (e.g., antibody or DVD-Ig) production, and, in particular, to methods and compositions for controlling and limiting the heterogeneity of proteins expressed in host cells. The production of proteins for biopharmaceutical applications typically involves the use of cell cultures that are known to produce proteins exhibiting varying levels of heterogeneity. The basis for such heterogeneity includes, but is not limited to, the presence of distinct glycosylation profiles in the produced proteins. For example, but not by way of limitation, such heterogeneity can be observed as an increase in high mannose N-glycans and NGA2F-GlcNAc species as well as a decrease in fucosylated species, such as NGA2F species.
The glycosylation profile of a protein (e.g., an antibody or DVD-Ig) can influence its biological activity through changes in half-life due to effects on clearance, folding, stability and antibody-dependent cellular cytotoxicity (ADCC) (Shental-Behor D. et al., (2008) PNAS 105:8256-8261; Kuhlmann M. et al., (2006) Nephrol. Dial. Transplant 21:v4-v8; Zheng K. et al., (2011) mAbs 3(6):568-576). ADCC is one mechanism responsible for the therapeutic effect of antibodies such as the anti-CD20 IgG1 rituximab and the anti-Her2/neu IgG1 trastuzumab. ADCC activity is influenced by the amount of fucose linked to the innermost GlcNAc of the Fc region, with enhanced activity seen with a reduction in fucose (Mori K. et al., (2007) Cytotechnology 55:109-114).
Heterogeneity of protein glycosylation can be assayed by releasing oligosaccharides present on the protein of interest (e.g., an antibody or DVD-Ig) via enzymatic digestion with, for example, N-glycanase. Once the glycans are released, the free reducing end of each glycan can be labeled by reductive amination with a fluorescent tag. The resulting labeled glycans are separated by normal-phase HPLC (NP-HPLC) and detected by a fluorescence detector for quantitation. Technological advances in recombinant protein production analysis have provided unique opportunities for identifying the extent of glycosylation exhibited by a particular protein population, particularly in the context of large-scale production of recombinant proteins.
Although such advances have allowed for the robust characterization of protein glycosylation, there remains a need in the art for culture conditions and production methods that allow for control over the glycosylation profile of a protein therapeutic. Modulation of protein glycosylation is particularly advantageous in the context of cell culture processes used for commercially produced recombinant bio-therapeutics as glycosylation has the potential to impact therapeutic utility. Control of the glycosylation profile of a therapeutic protein (e.g., an antibody or an antigen binding fragment thereof, or a DVD-Ig) is also critical for ensuring the production of comparable proteins such as biosimilars. Accordingly, there is a need in the art for compositions and methods for the targeted modulation of protein glycosylation. The instant invention addresses this need by providing compositions and methods for modulating protein glycosylation. The invention further provides methods for the targeted modulation of mannosylated and fucosylated N-glycan species linked to a protein of interest (e.g., antibody or DVD-Ig).